U.S. patent application number 15/887676 was filed with the patent office on 2018-08-02 for trap arrays for robust microfluidic sample digitization.
This patent application is currently assigned to University of Maryland, College Park. The applicant listed for this patent is University of Maryland, College Park. Invention is credited to Don L. DeVoe, Alex Sposito.
Application Number | 20180214873 15/887676 |
Document ID | / |
Family ID | 62977392 |
Filed Date | 2018-08-02 |
United States Patent
Application |
20180214873 |
Kind Code |
A1 |
DeVoe; Don L. ; et
al. |
August 2, 2018 |
TRAP ARRAYS FOR ROBUST MICROFLUIDIC SAMPLE DIGITIZATION
Abstract
A microfluidic device including at least one channel in fluid
communication with a sample trap array. Specifically, the
configuration and geometry of the trap arrays according to the
present invention allows for performing sample digitization that
supports passive self-discretization within the sample traps
without the need for any external flow control or actuation.
Geometrical parameters defining the sample traps, including the
trap width and the trap depth, are selected to optimize
self-filling of the sample traps. Reagents are incorporated into
the sample traps during device fabrication to allow for performing
multiplexed reactions within the sample traps.
Inventors: |
DeVoe; Don L.; (Bethesda,
MD) ; Sposito; Alex; (Arlington, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Maryland, College Park |
College Park |
MD |
US |
|
|
Assignee: |
University of Maryland, College
Park
College Park
MD
|
Family ID: |
62977392 |
Appl. No.: |
15/887676 |
Filed: |
February 2, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62453763 |
Feb 2, 2017 |
|
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|
62514501 |
Jun 2, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/50273 20130101;
B01L 2400/0688 20130101; B01L 2200/027 20130101; B01L 2300/0893
20130101; B01L 3/502753 20130101; B01L 2400/0406 20130101; B01L
2200/0605 20130101; B01L 7/52 20130101; B01L 2300/0864 20130101;
B01L 2300/0816 20130101; B01L 2300/0819 20130101; B01L 2300/0858
20130101; C12Q 1/686 20130101; B01L 2200/12 20130101; B01L
2300/0645 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; C12Q 1/686 20060101 C12Q001/686; B01L 7/00 20060101
B01L007/00 |
Claims
1. A self-loading microfluidic device comprising: a substrate
having at least one microfluidic channel provided therein, the
microfluidic channel being in communication with inlet and outlet
ports, wherein a sample is introduced into the microfluidic channel
through the inlet port; a plurality of sample traps branching off
the microfluidic channel; a capillary pump in fluid communication
with the channel and the outlet port, the capillary pump configured
to remove an excess sample from the microfluidic channel; a sealing
layer bonded to the substrate; and wherein each of the plurality of
traps has geometrical parameters selected to optimize self-filling
of the sample traps with the sample.
2. The microfluidic device of claim 1, wherein the geometrical
parameters include a channel width, a trap width, a trap depth, a
channel height, and a trap height.
3. The microfluidic device of claim 1, wherein the sample traps are
lateral protrusions branching off from the same side of the
microfluidic channel.
4. The microfluidic device of claim 1, wherein the sample traps are
lateral protrusions branching off from both sides of the
microfluidic channel.
5. The microfluidic device of claim 1, wherein the sample traps are
lateral protrusions branching off from both sides of the
microfluidic channel and are centerline offset.
6. The microfluidic device of claim 1, wherein the sample traps are
vertical traps beneath the microfluidic channel.
7. The microfluidic device of claim 1, wherein the sealing layer is
a thin film.
8. The microfluidic device of claim 7, wherein the thickness of the
thin film is 200 .mu.m.
9. The microfluidic device of claim 1, wherein each sample trap
comprises pre-deposited reagents.
10. The microfluidic device of claim 9, wherein each the
pre-deposited reagents are printed onto the sealing layer during
device fabrication.
11. The microfluidic device of claim 9, wherein the pre-deposited
reagents are polymerase chain reaction (PCR) reagents including
primers to amplify target nucleic acids in the sample, the PCR
reagents encapsulated into a paraffin wax ensuring that the primers
remain encapsulated during sample introduction.
12. The microfluidic device of claim 9, wherein the pre-deposited
reagents are loop-mediated isothermal amplification (LAMP) reagents
including primers to amplify target nucleic acids in the sample,
the LAMP reagents encapsulated into a paraffin wax ensuring that
the LAMP reagents remain encapsulated during sample
introduction.
13. The microfluidic device of claim 11, wherein the primers are
released by temperature application prior to thermocycling.
14. The microfluidic device of claim 1, wherein the capillary pump
is an absorbent membrane.
15. The microfluidic device of claim 14, wherein the absorbent
membrane is a polyvinylidene fluoride (PVDF) membrane.
16. The microfluidic device of claim 1, wherein the substrate is
fabricated from a thermoplastic polymer.
17. The microfluidic device of claim 1, wherein the substrate is
fabricated from cyclic olefin polymer (COP).
18. The microfluidic device of claim 1, wherein the sample is
injected into the inlet port by a pipette.
19. The microfluidic device of claim 1, wherein the trap has
dimensions of 900 .mu.m square and 250 .mu.m deep.
20. The microfluidic device of claim 1, wherein an immiscible phase
is loaded into the device to fully isolate each sample trap after
an excess sample is removed from the microfluidic channel by the
capillary pump.
21. The microfluidic device of claim 20, wherein the immiscible
phase is silicone oil.
22. A method comprising: providing a substrate having at least one
microfluidic channel provided therein, the microfluidic channel
being in fluid communication with inlet and outlet ports and a
plurality of sample traps branching off the microfluidic channel,
wherein the microfluidic channel connects the inlet port to a
capillary pump with the sample traps in between, wherein a sealing
layer is bonded to the substrate; introducing a sample into the
microfluidic channel through the inlet port; removing an excess
sample from the microfluidic channel by the capillary pump; filling
the microfluidic channel with an immiscible phase to isolate the
sample traps; and wherein each of the plurality of traps has
geometrical parameters selected to optimize self-filling of the
sample traps with the sample.
23. The method of claim 22, wherein the geometrical parameters
include a channel width, a trap width, a trap depth, a channel
height, and a trap height.
24. The method of claim 22, wherein the sample traps branch off
from the same side of the channel.
25. The method of claim 22, wherein the sample traps branch off
from both sides of the channel.
26. The method of claim 22, wherein the sample traps branch off
from both sides of the channel and are centerline offset.
27. The method of claim 22, wherein the microfluidic channel is a
planar channel and the sample traps are vertical traps beneath the
planar microfluidic channel.
28. The method of claim 22, wherein the sealing layer is a thin
film layer.
29. The method of claim 28, wherein the thickness of the thin film
layer is 200 .mu.m.
30. The method of claim 22, wherein each sample trap comprises
pre-deposited reagents.
31. The method of claim 30, wherein the pre-deposited reagents are
integrated into the microfluidic device during fabrication to
perform a different reaction within each sample trap.
32. The method of claim 22, wherein the pre-deposited reagents are
loop-mediated isothermal amplification (LAMP) reagents including
primers to amplify target nucleic acids in the sample, the LAMP
reagents encapsulated into a paraffin wax ensuring that the LAMP
reagents remain encapsulated during sample introduction.
33. The method of claim 22, wherein the pre-deposited reagents are
polymerase chain reaction (PCR) reagents including primers to
amplify target nucleic acids in the sample, the PCR reagents
encapsulated into a paraffin wax ensuring that the primers remain
encapsulated during sample introduction.
34. The method of claim 22, wherein the capillary pump is an
absorbent membrane.
35. The method of claim 34, wherein the absorbent membrane is a
polyvinylidene fluoride (PVDF) membrane.
36. The method of claim 22, wherein the sample is injected into the
inlet port by a pipette.
37. The method of claim 33, wherein the primers are released from
the paraffin wax by temperature application prior to
thermocycling.
38. The method of claim 22, wherein the trap has dimensions of 900
.mu.m square and 250 .mu.m deep.
39. The method of claim 22, wherein an immiscible phase is loaded
into the device to fully isolate each sample trap after an excess
sample is removed from the microfluidic channel by the capillary
pump.
40. The method of claim 22, wherein the immiscible phase is
silicone oil.
41. The method of claim 22, wherein the substrate is fabricated
from a thermoplastic polymer.
42. The method of claim 22, wherein the substrate is fabricated
from thermoplastic cyclic olefin polymer (COP).
43. A method for fabricating a self-loading microfluidic device,
the method comprising: providing a substrate; creating in the
substrate at least one microfluidic channel and microfluidic
features including a chamber, an inlet port, an outlet port, and
sample traps branching off the channel, wherein the microfluidic
channel connects the inlet port to the chamber with the sample
traps in between; inserting at least one absorbent membrane into
the chamber to remove an excess sample from the microfluidic
channel; mating the substrate to a sealing layer affixed to a
spotting stage to seal the at least one channel and the traps, the
sealing layer having reagents deposited thereon; and applying a
pressure to bond the sealing layer to the substrate.
44. The method of claim 43, further comprising depositing reagents
into the sample traps.
45. The method of claim 43, further comprising depositing reagents
to the sealing layer by a pin spotting tool through contact
printing prior to bonding the sealing layer to the substrate.
46. The method of claim 43, further comprising drying reagent spots
before a protective layer of a soft paraffin wax is deposited over
top of the dried reagent spots.
47. The method of claim 44, wherein the reagents comprise primers
and polyethylene glycol (PEG).
48. The method of claim 43, wherein the absorbent membrane is a
polyvinylidene fluoride (PVDF) membrane.
49. The method of claim 43, wherein the substrate is fabricated
from thermoplastic cyclic olefin polymer (COP).
50. The method of claim 43, wherein the traps branch off from both
sides of the channel and are centerline offset.
51. The method of claim 43, wherein the sample traps are lateral
protrusions branching off from the same side of the microfluidic
channel.
52. The method of claim 43, wherein the sample traps are lateral
protrusions branching off from both sides of the microfluidic
channel.
53. The method of claim 43, wherein the sample traps are vertical
traps beneath the microfluidic channel.
54. The method of claim 43, wherein the sealing layer is a thin
film layer.
55. The method of claim 54, wherein the thickness of the thin film
layer is 200 .mu.m.
56. The method of claim 55, wherein the trap has dimensions of 900
.mu.m square and 250 .mu.m deep.
57. The method of claim 43, wherein each of the plurality of traps
has geometrical parameters selected to optimize self-filling of the
sample traps with the sample, the geometrical parameters including
a channel width, a trap width, a trap depth, a channel height, and
a trap height.
58. A device comprising: a USB shell comprising: a self-loading
trap-array chip comprising: a substrate having at least one
microfluidic channel provided therein, wherein a sample is
introduced into the microfluidic channel through an inlet port; a
plurality of sample traps branching off the microfluidic channel,
each sample trap comprising reagents; a capillary pump in fluid
communication with the channel and an outlet port, the capillary
pump configured to remove an excess sample from the microfluidic
channel prior to filling the microfluidic channel with an
immiscible phase to isolate the sample traps; electrodes patterned
on a backside of the self-loading trap-array to sense the
temperature; a LED array in optical communication with the
self-loading trap-array; a CMOS imager to perform fluorescence
imaging of the trap array; and a routing board to control the
amplification of target nucleic acids in the biological sample.
59. The device of claim 58, wherein the microfluidic channel is a
planar channel and the sample traps are vertical traps beneath the
planar microfluidic channel.
60. The device of claim 58, wherein each sample trap comprises
pre-deposited reagents printed onto a sealing layer during device
fabrication.
61. The device of claim 60, wherein the pre-deposited reagents are
loop-mediated isothermal amplification (LAMP) reagents encapsulated
into a paraffin wax ensuring that the LAMP reagents remain
encapsulated during sample introduction.
62. The device of claim 58, wherein the capillary pump is an
absorbent membrane.
63. The device of claim 62, wherein the absorbent membrane is a
polyvinylidene fluoride (PVDF) membrane.
64. The device of claim 58, wherein the immiscible phase is
silicone oil.
65. The device of claim 58, wherein the substrate is fabricated
from thermoplastic cyclic olefin polymer (COP).
66. The device of claim 58, wherein the sample is injected into the
inlet port by a pipette.
67. The device of claim 62, wherein an excess sample is removed
from the channel by the absorbent membrane and then the immiscible
phase is loaded into the device to fully isolate each trap.
Description
INCORPORATION BY REFERENCE
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application Ser. Nos. 62/514,501, filed on Jun.
2, 2017, and 62/453,763, filed on Feb. 2, 2017, which are
incorporated herein by reference in their entireties.
[0002] The present application includes a Sequence Listing filed in
electronic format. The Sequence Listing is entitled
"3400-327_ST25.txt" created on Feb. 2, 2018, and is 2,000 bytes in
size. The information in the electronic format of the Sequence
Listing is part of the present application and is incorporated
herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention relates to a device for performing
passive microfluidic sample digitization without the need for any
external flow control or actuation. Furthermore, the device
according to the present invention can be used for multiplexed
amplification reactions.
Description of Related Art
[0004] Sample digitization, or the segregation of sample volume
into smaller parts, is an important operation required in many
applications for genomics, clinical diagnostics, and drug
discovery. The conventional approach to forming an array of
discrete fluid volumes from an initial sample solution has been to
rely on robotic fluidic handling. However, this approach requires
cumbersome and costly equipment, suffers from unfavorable scaling
in multistep assays, and is generally restricted to discretized
sample volumes in the microliter range. Moreover, the need for an
open substrate such as a microwell plate for deposition increases
the risk of external contamination, introduces the need to limit
sample evaporation, and constrains the types of assay operations
that may be performed.
[0005] A variety of microfluidic technologies have been developed
to enable automated sample digitization within enclosed flow
systems. One of the most common approaches to microfluidic
digitization is droplet generation, an active digitization process
wherein a sample volume is dispersed within an immiscible phase to
create small uniform reactors defined by individual droplets.
Microfluidic droplet generators allow the flow rates of the
continuous and dispersed phases to be adjusted for control over the
volume and production rate of the digitized sample volumes, and can
be readily used for the formation of high density arrays. However,
droplet generation is an active digitization method requiring
continuous and precise flow control for monodispersed droplet
formation, necessitating the use of fluidic interfacing and flow
control hardware, thereby increasing the complexity and cost of the
final device. Furthermore, because of the active nature of the
droplet generation process, the resulting droplets typically
require additional mechanisms downstream for manipulation and assay
analysis. While a range of methods for downstream control via
droplet trapping and release using hydrodynamic, by optical or
acoustic manipulation have been explored, these added operations
can degrade the potential for simplicity, affordability, and
integration offered by microfluidics.
[0006] Electrowetting-on-dielectric (EWOD) represents an
alternative active digitization technique that enables on-demand
formation of discrete sample volumes together with controlled
manipulation of individual droplets for subsequent assay
operations. In the EWOD technology, differential capillary forces
are generated across a droplet by controlling the surface contact
angle between the droplet and an underlying substrate through
application of an external electric potential, allowing sample
packets to be segregated and transported by direct voltage control.
Despite this unique functionality, EWOD devices can present
challenges in scalability related to electrode addressing, demand
high voltages for operation, and require relatively complex
fabrication methods to define both the dielectric and electrode
layers needed for reliable device operation.
[0007] Driven by the need for simpler and more robust methods of
sample discretization, a number of passive digitization methods
have been developed. Passive sample digitization takes advantage of
processes that do not require precise control over fluid flow or
the use of active control elements, such that discrete volumes are
created on-chip automatically within spatially indexed locations. A
central advantage of these passive methods over active digitization
is that the instrumentation required for compartmentalization is
greatly reduced or eliminated, making these techniques very well
suited for use in devices where low cost and simple operation are
important considerations.
[0008] The passive sample digitization concept has been
successfully applied to various open fluidic platforms in which
sample is discretized within arrays of patterned microwells by
sequential well priming and selective dewetting of the surrounding
field while leaving individual fluid volumes anchored within the
wells. Similarly, selective patterning of hydrophilic regions or
porous absorbent materials within wells on a hydrophobic surface
has been employed to initiate wetting in specific locations while
allowing excess sample to be easily removed.
[0009] A related method has been applied to sealed microfluidic
systems, allowing the passive discretization of sample in enclosed
microchannels. While various device geometries have been explored,
they share a similar approach in which sample is introduced through
a microchannel by pressure driven flow, vacuum, or centrifugal
actuation to prime a series of wells fluidically connected to one
of the microchannel walls, followed by the introduction of an
immiscible oil phase to remove residual sample from the
microchannel. The oil flow serves to shear off sample from the
filled wells, leaving digitized aqueous fluid volumes behind. The
oil phase used to backfill the chip also serves to fully isolate
the sample volumes and prevent evaporation. In these devices,
polydimethylsiloxane (PDMS) is commonly chosen as a substrate
material due to its high air permeability, enabling dead-end
filling of the wells without trapping air bubbles during priming.
The devices are also typically filled with an oil phase prior to
sample introduction, thereby enhancing the filling of aqueous
sample into hydrophobic PDMS wells, and improving sample retention
during the final oil backfill. Accordingly, a new approach to
sample digitization that supports passive self-discretization
without any external flow control or actuation is needed.
[0010] Multiplex PCR (mPCR) is an approach to increasing the
throughput of nucleic acid analysis by allowing the amplification
and detection of multiple sequence targets in a single PCR
reaction. For example, mPCR has been widely applied to the analysis
of antibiotic-resistance genes from bacterial samples, with a range
of mPCR assays developed for identification of antibiotic-resistant
enterobacteriaceae, enterococci, Vibrio cholerae, and S. aureus.
However, complexity of primer design and validation for mPCR
remains a significant limitation for new assay development. More
fundamentally, primer competition constrains multiplexing depth,
with 5-plex assays representing the nominal maximum, and spectral
overlap of fluorescent probes used in real-time PCR (qPCR) further
limits the number of amplicons that may be detected in a single
reaction. A sequential mPCR approach involving a series of
individual microliter-volume mPCR reactions using different primer
sets has recently emerged, but at the expense of assay time.
[0011] As an alternative to mPCR, multiplexing may be achieved by
performing multiple individual PCR reactions in an array of
spatially-isolated reaction wells. Commercially available microwell
plate platforms supporting multiplexed PCR with integrated reagents
have been developed, but are limited by high consumable costs and
the need for complex instrumentation for system operation. Several
microfluidic technologies have been explored to address these
constraints, by reducing reagent and infrastructure requirements
while enabling semi-automated assay operation. Approaches include
the use of centrifugal microfluidics where centrifugal actuation is
used to actively deliver sample to on-chip reaction chambers
containing specific PCR primer sets, and reconfigurable
microfluidic devices where isolated primer sets on one substrate
are combined with individual reaction chambers on a second
substrate through relative motion between the chip elements.
However, these approaches still rely on significant supporting
equipment for operation.
[0012] Accordingly, there is a need for a low cost microfluidic
platform designed to perform highly scalable multiplexed PCR with
minimal manual input.
SUMMARY OF THE INVENTION
[0013] The present invention relates to microfluidic devices
including a sample trap (also referred to as sample well) array.
Specifically, the configuration and geometry of the trap arrays
according to the present invention allows for performing sample
digitization that supports passive self-discretization within the
sample traps without the need for any external flow control or
actuation.
[0014] In one aspect of the invention, a self-loading microfluidic
device and a method for loading the microfluidic device are
provided. Specifically, the microfluidic device comprises a
substrate having at least one microfluidic channel provided
therein. In one embodiment, the substrate is fabricated from a
thermoplastic polymer. The microfluidic channel is in fluid
communication with inlet and outlet ports. A sample is introduced
into the microfluidic channel through the inlet port. Furthermore,
the microfluidic device includes a plurality of sample traps
branching off the microfluidic channel. Each of the plurality of
traps has geometrical parameters selected to optimize loading of
the sample traps with the sample. A capillary pump in fluid
communication with the microfluidic channel is configured to remove
an excess sample from the microfluidic channel. An immiscible phase
is loaded into the microfluidic channel to fully isolate each
sample trap after an excess sample is removed from the microfluidic
channel. A sealing layer is bonded to the thermal substrate.
Furthermore, each sample trap includes reagents integrated into the
microfluidic device during fabrication to perform a different
reaction within each sample trap.
[0015] In yet another aspect of the present invention, a method for
fabricating a self-loading microfluidic device is provided.
Specifically, at least one microfluidic channel and microfluidic
features are provided in a substrate. In one embodiment, the
substrate is fabricated from a thermoplastic polymer. The
microfluidic features include a chamber, an inlet port, an outlet
port, and sample traps branching off the microfluidic channel. The
microfluidic channel connects the inlet port to the chamber with
the sample traps positioned in between. Next, at least one
absorbent membrane is inserted into the chamber to remove an excess
sample from the microfluidic channel. The thermoplastic substrate
is mated to a sealing layer affixed to a spotting stage to seal the
at least one microfluidic channel and the sample traps. In one
embodiment, reagents are deposited on the sealing layer. Finally, a
pressure is applied to bond the sealing layer to the substrate. In
one embodiment, the method further comprises depositing reagents to
the sealing layer prior to bonding it to the thermoplastic
substrate. By way of example and without limitation, the reagents
may be deposited to the sealing layer by a pin spotting tool
through contact printing.
[0016] In yet another aspect of the invention, a device including a
USB shell is provided. The USB shell comprises a self-loading chip
having a sample trap array, electrodes patterned on a backside of
the self-loading chip to sense the temperature, a LED array in
optical communication with the self-loading trap array, a CMOS
imager to perform fluorescence imaging of the trap array, and a
routing board to control the amplification of target nucleic acids
in a biological sample.
[0017] The self-loading chip comprises a substrate having at least
one microfluidic channel provided therein. In one embodiment, the
substrate is fabricated from a thermoplastic polymer. A sample is
introduced into the microfluidic channel through an inlet port. A
plurality of sample traps comprising reagents branches off the
microfluidic channel. A capillary pump is in fluid communication
with the channel and an outlet port. The capillary pump is
configured to remove an excess sample from the microfluidic channel
prior to filling the microfluidic channel with an immiscible phase
to isolate the sample traps.
[0018] In one embodiment, each sample trap includes reagents
integrated into the microfluidic device during fabrication to
perform a different biological reaction within each sample trap. In
one embodiment, the reagents are encapsulated into a paraffin wax
ensuring that the reagents remain encapsulated during sample
loading. The primers are released from the paraffin wax by
temperature application prior to performing the biological
reaction.
[0019] In one embodiment of the present invention, the sample traps
are lateral protrusions branching off from the same side or from
the both sides (with or without centerline offset) of the
microfluidic channel. In yet another embodiment, the sample traps
are vertical traps beneath the microfluidic channel. In one
embodiment, the microfluidic features, including a channel,
inlet/outlet ports, sample traps, and a chamber, are milled in the
thermoplastic substrate. Alternatively, the microfluidic features
may be created on the thermoplastic substrate in a two-step
embossing process.
[0020] By way of example and without limitation, the sample trap
may have dimensions of 900 .mu.m square and 250 .mu.m deep. In yet
another embodiment, the sealing layer is a thin film, which in one
embodiment has a thickness of 200 .mu.m.
[0021] In one embodiment, the capillary pump is a fabricated
absorbent membrane, which in one embodiment is fabricated from a
polyvinylidene fluoride (PVDF). The substrate may be fabricated
from cyclic olefin polymer (COP). The sample may be injected into
the inlet port by a pipette.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0023] The accompanying drawings, which are incorporated herein and
form part of the specification, illustrate various embodiments of
the subject matter of this disclosure. In the drawings, like
reference numbers indicate identical or functionally similar
elements. Additionally, the left-most digit(s) of a reference
number identifies the drawing in which the reference number first
appears.
[0024] FIG. 1A illustrates a workflow and structural elements of a
self-loading and digitizing (SLD) device.
[0025] FIG. 1B illustrates a single sided trap configuration on an
SLD device.
[0026] FIG. 1C illustrates a double sided trap configuration on an
SLD device.
[0027] FIG. 1D illustrates a staggered trap configuration on an SLD
device.
[0028] FIG. 2A illustrates a sample loading process on an SLD
device having a single sided trap configuration.
[0029] FIG. 2B illustrates a geometric representation for the
single sided trap configuration at the instant the flow front
touches the opposite trap wall at varying advancing front angle
.theta.a.
[0030] FIG. 2C illustrates trap aspect ratio (TAR) dependence on
advancing front angle .theta.a for the SLD device of FIG. 2A having
a single sided trap configuration.
[0031] FIG. 2D illustrates TAR dependence on fluidic aspect ratio
(FAR) for the SLD device of FIG. 2A having a single sided trap
configuration.
[0032] FIG. 2F is a geometric model for a single sided trap
design.
[0033] FIG. 3A illustrates a sample loading process for an SLD
device having a double sided trap configuration.
[0034] FIG. 3B illustrates a geometric representation for the
double sided trap configuration at the instant the front touches
the opposite trap wall at varying advancing front angle
.theta.a.
[0035] FIG. 3C illustrates TAR dependence on advancing front angle
.theta.a for the double sided SLD device.
[0036] FIG. 3D illustrates TAR dependence on FAR for the double
sided SLD device.
[0037] FIG. 3F is a geometric model for a double sided trap
design.
[0038] FIG. 4A illustrates a loading process for an SLD device
having a staggered trap configuration.
[0039] FIG. 4B illustrates a geometric representation for the
staggered trap configuration at the instant the flow front touches
the opposite trap wall at varying advancing front angle
.theta.a.
[0040] FIG. 4C illustrates TAR dependence on advancing front angle
.theta.a for the SLD device of FIG. 4A having a staggered trap
configuration.
[0041] FIG. 4D illustrates TAR dependence on FAR for the SLD device
of FIG. 4A having a staggered trap configuration.
[0042] FIG. 4F is a geometric model for a staggered trap
design.
[0043] FIG. 5 illustrates the sample loading percentage for
different trap configurations at different aspect ratios. Two data
sets (light and dark) for each trap configuration represent a
difference in heights between the main channel and the trap.
[0044] FIG. 6A illustrates the design space for the single sided
configuration.
[0045] FIG. 6B illustrates the design space for the double sided
configuration.
[0046] FIG. 6C illustrates the design space for the staggered
configuration.
[0047] FIG. 7A is an image of an SLD chip having a trap array
according to one embodiment of the present invention.
[0048] FIG. 7B is a histogram of volume distribution in the loaded
SLD device of FIG. 7A.
[0049] FIG. 7C illustrates an SLD chip having a trap array
according to another embodiment of the present invention.
[0050] FIG. 8A illustrates an image and schematic for an SLD chip
according to one embodiment of the present invention.
[0051] FIG. 8B is a flowchart illustrating fabrication process of
an SLD chip as shown in FIG. 8A.
[0052] FIG. 9 illustrates an image sequence of PEG and fluorescein
salt being rehydrated by sample self-loading in the SLD chip of
FIG. 8A.
[0053] FIG. 10A is an image of a dried down PEG/primer spot.
[0054] FIG. 10B is an image of a PEG/primer spot covered by a
paraffin wax capping layer.
[0055] FIG. 10C is an image of primer spots capped with paraffin in
the SLD chip of FIG. 8A.
[0056] FIG. 11 is an image sequence demonstrating a temperature
controlled release of fluorescein salt mixed with primer and PEG
printing solution.
[0057] FIG. 12A illustrates an experimental design of the two step
multiplex assay on the SLD platform.
[0058] FIG. 12B is a table of experimental results with pUC19 and
pBR322 template loaded chips.
[0059] FIGS. 13A-13C illustrate a pipette-based sample preparation
decoupling the purification and concentration of nucleic acids from
the trap chip operation.
[0060] FIG. 14 illustrates filling process for an SLD device having
vertical traps.
[0061] FIGS. 15A-15B illustrate comparison of the staggered trap
design (FIG. 15A) to the planar vertical trap design (FIG.
15B).
[0062] FIG. 16 illustrates a loop-mediated isothermal amplification
(LAMP) reaction scheme.
[0063] FIG. 17A illustrates a CMOS imager positioned below a trap
chip.
[0064] FIG. 17B illustrates contact imaging results using
fluorescent dye.
[0065] FIG. 18 illustrates a reusable CMOS contact fluorescence
system integrated into a USB stick.
DETAILED DESCRIPTION
[0066] The present invention is directed to sample digitization
that supports passive self-discretization without the need for any
external flow control or actuation. The technique exploits
controlled pinning of fluid at geometric discontinuities within a
microchannel, and may be applied to devices manufactured from any
material with defined surface properties, including thermoplastics.
Specifically, the present technique enables digitization of an
aqueous sample in a thermoplastic cyclic olefin polymer (COP)
microfluidic device, taking advantage of the polymer's moderate
surface energy to achieve fully passive self-discretization without
the need for any external pumps or other flow control elements.
[0067] Furthermore, an analytic model was used for predicting the
maximum ratio of trap depth to trap width at which complete trap
filling will occur. The model was validated through an experimental
evaluation of the filling process using a set of devices fabricated
with parametrically-varying trap geometries. Finally, the model was
used as a predictive tool for the design and fabrication of a high
aspect ratio staggered trap array, allowing the reliability of the
filling process to be evaluated in a high density format.
[0068] FIG. 1A is an illustration of workflow in a self-loading and
digitizing (SLD) device 100 comprising an inlet port 116, a main
microfluidic channel 102, sample traps 104, a capillary pump 106,
and an outlet port 112. The sample traps 104 are lateral
protrusions emerging from the sides of the main microfluidic
channel 102. A sample solution 110 is received at the inlet port
116 to flow along the main channel 102 filling the sample traps
104. In one embodiment, the sample solution 110 is delivered to the
inlet port 116 by a pipette 108. To purge excess sample from the
main channel 102, the main channel 102 terminates into a large
chamber 118 ending with the outlet port 112. The chamber 118 has
integrated capillary pump 106 so that the main channel 102 can be
backfilled with an immiscible phase for reducing sample
evaporation, as well as encapsulating each digitized sample volume
with an immiscible barrier to prevent unwanted sample
contamination. In one embodiment, the capillary pump is in the form
of an absorbent membrane. By way of example and without limitation,
the absorbent membrane may be fabricated from hydrophilically
modified polyvinylidene fluoride (PVDF). The sample traps 104
reside on the side of the main channel 102 to provide the greatest
flexibility in designing of the trap geometry. In one embodiment,
the SLD device 100 is constructed with thermoplastic polymer and
loaded with an aqueous test solution with surfactant. By way of
example and without limitation, the thermoplastic polymer may be
cyclic olefin polymer (COP), a weakly hydrophobic polymer.
[0069] As further demonstrated in FIG. 1A, the sample 110 is
received at the inlet port 116, step 122. Next, the sample 110
advances along the main channel 102 filling the traps 104, step
124. In step 126, an excess sample is absorbed by the capillary
pump 106 leaving the main channel 102 empty (step 128). Next, in
step 130, the main channel 102 is loaded with an immiscible phase
120 to fully isolate each sample trap 104. In one embodiment, the
immiscible phase 120 may be oil.
[0070] The angle of the advancing sample front .theta.a (FIGS. 2F,
3F, and 4F) is an important factor in the loading physics of the
SLD device 100 and is a function of the surface interactions
between the sample solution and substrate, as well as the effect of
gravity on the fluid head in the inlet. In one embodiment, to
achieve self-loading, a small amount of surfactant (by way of
example and without limitation 0.06% w/w) was added to the sample
solution. Each trap 104 is characterized by a trap aspect ratio
(TAR) and fluidic aspect ratio (FAR) defined with reference to
FIGS. 2B, 3B, and 4B. Specifically, TAR=f.sub.y/w.sub.t and
FAR=w.sub.c/w.sub.t, where w.sub.c is the main channel width,
w.sub.t is the trap width, and f.sub.y is a distance the sample
traveled into the trap.
[0071] For example, if the sample traps 104 were intended to be
pre-spotted with assay reagents and digitized volumes were expected
to remain discrete, the TAR could be designed such that the depth
of the trap is greater than it takes for reagents to diffuse into
the main sample flow during loading, which would cause
contamination of downstream traps. Furthermore, the TAR determines
the percentage of volume retained for analysis versus the amount of
sample required to prime the device.
[0072] As demonstrated in FIG. 1A, approximately the volume of the
main channel is lost because it is required to drive the sample
front to the capillary pump (PVDF membrane) 106. The sample excess
is wicked away leaving the main channel 102 for the immiscible
phase (oil) 120 backfill. Moreover, due to the physics of loading
via surface tension the ratio of trap height (d.sub.t) to main
channel height (d.sub.c) cannot exceed 1 because in that case the
advancing front would favor bypassing the trap due to the
interfacial expansion required for it to enter. The trap height
(d.sub.t) and the main channel height (d.sub.c) refer to the
out-of-plane dimension for the main channel 102 and traps 104.
[0073] Finally, trap configuration with respect to one another is
an important factor in a surface tension driven system as it
contributes the front conformation during trap loading which
ultimately contributes to the types of trap geometries and more
specifically the trap aspect ratios that can be loaded
effectively.
[0074] The spatial positioning of the sample traps 104 can be
broken down in three distinct configurations: traps all along one
side of the main channel 102 (single sided (SS)) as shown in FIG.
1B; traps on both sides of the main channel 102 directly adjacent
(double sided (DS)) as shown in FIG. 1C; traps that are on both
sides of the main channel 102, but centerline offset from one
another (staggered (S)) as shown in FIG. 1D. FIG. 1A demonstrates
the traps 104 in the staggered configuration.
[0075] In one embodiment, the SLD devices according to FIG. 1A were
fabricated by providing channel and microwell features in a plaque
using a computer numerical controlled 3-axis CNC machine. By way of
example and without limitation, the plaque may be a COP plaque. A
hydrophilically modified polyvinylidene fluoride (PVDF) absorbent
membrane was cut to specific size. The plaque was immersed in a
solution of decahydronaphthalene in ethanol, rinsed with ethanol,
and blown dry with N.sub.2. The absorbent membrane was then
manually aligned to a chamber in the plaque before a sealing layer
was pressed to the substrate in a hot press to complete the
bonding. In one embodiment, the sealing layer is a thin film. By
way of example and without limitation, the thickness of the thin
film may be about 200 .mu.m.
[0076] FIG. 2A demonstrates images taken during sample loading
process for an SLD device having a single sided trap configuration
(FIG. 1B). The single sided configuration consists of the traps 104
branching off the same side of the main channel 102. The loading
process for this configuration as shown in FIG. 2B starts when the
sample flow reaches the entrance of the trap. The side of the
sample front that faces the trap will be pinned due to the change
in geometry while surface energy will drive the other side of the
advancing front along the main channel wall. Once, the critical
angle for advancement is reached at the pining point the front
begins to spread into the trap. Surface tension will stabilize the
interface so that angle of the advancing front (.theta.a) is equal
on both sides of the interface. The advancing front will eventually
make contact with the wall on the opposite side of the trap. At
this point the front can no longer fill the trap because it has
trapped air ahead of it. The conformation of the front when it
touches the opposite wall will determine the distance it traveled
into trap (f.sub.y). After the front touches the opposite trap wall
surface tension will force it to regain its unpinned contact angle
.phi. on both sides of the trap, and this is true for all
configurations. The unpinned contact angle .phi. is a contact angle
of water on the thermoplastic surface which is a property of the
substrate material and can be measured for any given substrate.
[0077] FIGS. 2B and 2F demonstrate a geometric representation for
the single sided configuration at the instant the front touches the
opposite trap wall at varying .theta.a. The geometric model
presented in FIG. 2F illustrates the relationship that .theta.a,
and fluidic aspect ratio (FAR) will have on the final conformation
of the interface when it touches the opposite wall in the single
sided configuration. From the geometric model of FIG. 2F, an
analytical solution is derived using a set of equations and
constrains to solve for each point where the interface makes
contact with the wall as a way to predict the maximum TAR
parametrically. The calculations are performed under the assumption
of constant interface curvature. Referring to FIG. 2F, x.sub.o,
y.sub.0, y.sub.a, and r are unknown; x.sub.a=0, x.sub.b=w.sub.t,
x.sub.c=y.sub.a, y.sub.b=w.sub.c, y.sub.c=0, .phi. and .theta. are
known. Furthermore, y.sub.a.gtoreq.w.sub.c and r.sub.I.gtoreq.0.
Equations (1)-(4) describe the geometry as presented in FIG.
2F.
x 0 2 + ( y a - y 0 ) 2 = r I 2 ( 1 ) ( w t - x 0 ) 2 + ( w c - y 0
) 2 = r I 2 ( 2 ) ( y a - x 0 ) 2 + y 0 2 = r I 2 ( 3 ) tan .PHI. =
x o y a - y 0 ; .PHI. = .theta. a - .pi. / 2 ( 4 ) ##EQU00001##
Equations (1)-(4) are solved for f.sub.y=y.sub.a-w.sub.c.
[0078] TAR(f.sub.y/w.sub.t) dependence on .theta.a is plotted in
FIG. 2C for an experimental SLD device with dimensions w.sub.t=390
.mu.m and w.sub.c=200 .mu.m. The .theta.a range is defined by a
lower limit of 45.degree. because below this point the interface
will have negative curvature and thus is not able to be defined by
the system of equations where radial curvature is assumed positive
and equal at each point. In a physical sense .theta.a<45.degree.
indicates high wettability between the sample and the substrate, so
loading of high aspect ratios is not a concern. The maximum
achievable TAR with these parameters is 1:1 at a
.theta.a=45.degree.. As .theta.a.fwdarw.180.degree. the TAR
asymptotically approaches 0 because f.sub.y.fwdarw.w.sub.c.
[0079] TAR dependence on the FAR(w.sub.c/w.sub.t) is plotted in
FIG. 2D for an SLD device using a fixed .theta.a=90.degree.,
representing experimental conditions. The FAR range explored had an
upper limit of five because at these aspect ratios the sacrificed
sample volume required to load the traps would significantly
outweigh retained sample--sample efficiency of 3.8%. This plot
shows the highest achievable TAR is 1:1 as FAR.fwdarw.0, which
decays to 0 as FAR.fwdarw..infin.. The optimal design for this
configuration is to minimize the FAR and achieve a TAR close to 1:1
and a theoretical sample efficiency of 99.7%.
[0080] FIG. 3A demonstrates images taken during the loading process
for a double sided SLD. The double sided configuration shows traps
104 branching off from both sides of the main channel 102. The
loading process for this trap arrangement starts when the sample
flow reaches the entrance of the trap. The interface is pinned at
both sides of the bifurcation into the traps until .theta.a is
reached at which point the front will begin spreading down the trap
walls. It is possible that the interface will be asymmetric if
.theta.a is reached at different times due to the traps not being
perfectly aligned, or pinning points not being a perfect 90.degree.
corner. This asymmetry will cause instability in the interface that
will result in different travel distances into each of the traps.
Eventually the advancing front will make contact with the wall on
the opposite side of the trap and halt trap filling because of the
air pocket formed ahead of it. Similar to the single side model,
the conformation of the front when it touches the opposite wall
will determine the distance it traveled into trap.
[0081] The geometric model presented in FIGS. 3B and 3F illustrates
the relationship that .theta.a, and FAR will have on the final
conformation of the interface when it touches the opposite wall in
the double sided configuration. From this geometric model of FIG.
3F an analytical solution is derived using a set of specific
equations and constrains to solve for each point where the
interface makes contact with the wall as a way to predict the TAR
parametrically. The calculations are performed under assumptions of
constant interface curvature and symmetric advancement of
interfaces along both trap walls. Referring to FIG. 3F, element
302, x.sub.o, y.sub.0 y.sub.a, and r are unknown; x.sub.a=0,
x.sub.b=w.sub.t, y.sub.c=0, y.sub.b=w.sub.0/2, y.sub.0=0, .phi. and
.theta. are known. Furthermore, y.sub.a.gtoreq.w.sub.c/2,
r.sub.I.gtoreq.0, and .theta..sub.lim(FAR). Equations (1)-(3)
describe the geometry as presented in FIG. 3F, element 302.
x o 2 + y a 2 = r I 2 ( 1 ) ( w t - x o ) 2 + ( w c / 2 ) 2 = r I 2
( 2 ) tan .PHI. = x o y a ; .PHI. = .theta. a - .pi. / 2 ( 3 )
##EQU00002##
[0082] Equations (1)-(3) are solved for f.sub.y=y.sub.a-w.sub.c/2.
Referring to FIG. 3F, element 304, as the curvature is constant,
x.sub.o=w.sub.t/2 and y.sub.a=w.sub.c/2;
tan .PHI. lim = w t w c ##EQU00003##
and .theta..sub.lim=.phi..sub.lim+.pi./2.
[0083] TAR dependence on .theta.a is plotted in FIG. 3C for an SLD
device with dimensions w.sub.t=390 .mu.m and w.sub.c=200 .mu.m to
match experimental devices. Unlike the SS model, the .theta.a does
not have a lower limit within the DS model but nevertheless is
constrained to .theta.a>45.degree. because of the wettability of
the sample solution. The upper limit of .theta.a will be a function
of the FAR and is derived in FIG. 3F. For the experimental device
with wt=390 .mu.m and we=200 .mu.m the upper limit of
.theta.a=152.degree.. At .theta.a=45.degree. the TAR is
approximately 2.2 and as .theta.a is increased the TAR decays 0
when .theta.a=152.degree..
[0084] TAR dependence on the FAR is plotted in FIG. 2D for an
experimental SLD device assuming a fixed .theta.a=90.degree.,
representing experimental conditions. The FAR range explored had an
upper limit of 5 because at these aspect ratios the sacrificed
volume required to load sample would significantly outweigh
retained volume--sample efficiency of 13.8%. This plot shows the
highest achievable TAR is 1:1 as FAR.fwdarw.0, which decays to 0 as
FAR.fwdarw..infin.. The optimal design for this configuration is to
minimize the FAR to achieve a TAR close to 1:1 and a theoretical
sample efficiency of 99.9%.
[0085] FIG. 4A illustrates images taken during the loading process
for a staggered SLD device. The staggered configuration shows the
arrangement of traps 104 branching off from both sides of the main
channel 102 but instead of being aligned directly opposite they are
centerline offset.
[0086] The loading process for the staggered trap arrangement, as
shown in FIG. 4A, starts when the sample flow reaches the entrance
of the trap. The interface is pinned at the opening while the other
side of the front is able to traverse the main channel. Flow will
continue along the main channel wall until it gets pinned on the
opposite side trap entrance. When the front is pinned on both sides
the interface will expand and allow the critical angle for
advancement to be reached at the first pinning point. The front
will then begin to spread down the first trap wall and eventually
make contact with the wall on the opposite side of the trap and
halt trap filling because of the air pocket formed ahead of it. The
conformation of the front when it touches the opposite wall will
determine the distance it traveled into trap.
[0087] The geometric model presented in FIGS. 4B and 4F illustrates
the relationship that .theta.a, FAR, and pinning offset ratio (POR)
will have on the final conformation of the interface when it
touches the opposite wall in the staggered configuration. The
pinning offset (PO) is defined as the distance that separates the
entrance to the opening of the trap to the pinning point
established by the opening to the trap opposite of it. The POR is
just the PO normalized to trap width (w.sub.t). The PO is different
on each side of the main channel because on the staggered side the
pinning offset will include a barrier wall. So the pinning offset
in the staggered row PO*=(w.sub.t-PO)+w.sub.b. From the geometric
model of FIG. 4F, element 402, an analytical solution is derived
using a set of specific equations and constrains to solve for each
point where the interface makes contact with the wall as a way to
predict the TAR parametrically. The calculations are performed
under the assumption of constant interface curvature. Referring to
FIG. 4F, element 402, the calculations are made under the
assumption of constant interface curvature. Variables x.sub.o,
y.sub.0, y.sub.a, and r are unknown; x.sub.a=0, x.sub.b=w.sub.t,
x.sub.c=p, y.sub.b=y.sub.0, y.sub.c=0, .phi. and .theta. are known.
Furthermore, y.sub.a.gtoreq.w.sub.c, y.sub.0.gtoreq.w.sub.c, and
r.sub.I.gtoreq.0. Equations (1)-(4) describe the geometry as
presented in FIG. 4F, element 402.
x o 2 + ( y a - y 0 ) 2 = r I 2 ( 1 ) ( w t - x o ) 2 = r I 2 ( 2 )
( p - x o ) 2 + y 0 2 = r I 2 ( 3 ) tan .PHI. = x o y a - y 0 ;
.PHI. = .theta. a - .pi. / 2 ( 4 ) ##EQU00004##
Equations (1)-(4) are solved for f.sub.y=y.sub.a-w.sub.c.
[0088] For FIG. 4F, element 404, calculations are made under the
assumption of constant interface curvature. Variables x.sub.o,
y.sub.0, y.sub.a, and r are unknown; x.sub.a=0, x.sub.b=w.sub.t,
y.sub.a=p.sub.lim (p.sub.lim is also referred to as pinning offset
(PO)), y.sub.c=0, .phi. and .theta. are known. Furthermore,
y.sub.a.gtoreq.w.sub.c, and r.sub.I.gtoreq.0. Equations (1)-(4)
describe the geometry as presented in FIG. 4F, element 404.
x o 2 + ( p lim - y 0 ) 2 = r I 2 ( 1 ) ( w t - x o ) 2 + ( w c - y
o ) 2 = r I 2 ( 2 ) ( p lim - x o ) 2 + y 0 2 = r I 2 ( 3 ) tan
.PHI. = x o p lim - y 0 ( 4 ) ##EQU00005##
Equations (1)-(4) are solved for p.sub.lim(TAR, .theta.a) using the
single sided model. If p>p.sub.lim, then pinning does not occur
for that configuration.
[0089] TAR dependence on .theta.a is plotted in FIG. 4C for an
experimental SLD device with dimensions wt=390 .mu.m and we=200
.mu.m and POR=0.55. Similar to the DS model, the .theta.a does not
have a lower limit but is constrained to .theta.a>45.degree.
because of the wettability of the sample solution. At
.theta.a=45.degree. the TAR is approximately 3.7 for test device,
and as .theta.a is increased the TAR decays to 0 when
.theta.a=180.degree..
[0090] TAR dependence on the FAR is plotted in FIG. 4D for an
experimental SLD device using a fixed .theta.a=90.degree. with
varying PORs. The FAR range explored had an upper limit of 1
because of the model . . . . This plot shows the highest achievable
TAR of 2:1 as FAR.fwdarw.0 which decays linearly to 1 as the
FAR.fwdarw.1. The optimal design for this configuration is to
minimize the FAR and the POR to achieve a TAR close to 2:1 and a
theoretical sample efficiency of 99.9%.
[0091] FIG. 4D also plots single sided (SS) and double sided (DS)
models for comparison. The TAR DS and TAR SS will have the same
limit of 1 as FAR.fwdarw.0, while the TAR DS decays to 0 at a
lesser rate than TAR SS as the FAR.fwdarw..infin.. As the POR
approaches a limit, a function of the FAR (See FIG. 4F), the TAR S
dependence acts as if it were a single sided configuration. This
can be explained physically because at sufficiently high POR the
front will touch the opposite trap wall before it can reach the
pinning point and succumb to its affects. Overall, the staggered
model predicts a drastic improvement over both the double sided and
single sided configurations for filling aspect ratios greater than
1. Furthermore, the staggered configuration enables control over
diffusion lengths of pre-deposited reagents within traps mitigating
risk of cross contamination, a necessity for certain
applications.
[0092] Prior reported digitization platforms commonly initialize
operations with an oil phase plus surfactant which inhibits
sample/substrate interaction making digitization effectiveness
highly dependent on the control over pressure driven flow. On the
other hand, by eliminating the oil initialization step and instead
harnessing the surface energy of the substrate, it is not only
possible to eliminate unnecessary workflow but also leverage
surface energy to improve digitization effectiveness.
[0093] FIG. 5 illustrates loading percentage (%) of the different
trap configurations at different aspect ratios. Two data sets
(light and dark) for each trap configuration represent a difference
in heights between the main channel and the trap. There are several
important considerations such as interfacial instability caused by
alignment asymmetry, non-sharp pinning points, and surface
roughness that may cause a deviation from the model in experimental
SLD devices. In practice, these effects result in a gradient in
terms of loading percentage decay around the predicted TAR limit as
opposed to a sharp drop off. Another important distinction is that
the traps have curvature at the top which changes the interface
dynamics when it traverses the curvature in such a way that it is
difficult to incorporate into the model. It is not sufficient to
just assume filling will be spontaneous once it gets to the curved
portion because at the limit the interface will be unstable and
also will be very close to the opposite wall. To address this, the
TAR, as defined in the experimental devices, includes the curvature
at the top as opposed to where the curvature starts because filling
is assumed to be spontaneous after that point. This will push the
loading percentage limit to above the theoretical limit predicted
by the model to account for the improved but not absolute filling
in the curved section.
[0094] In one embodiment, 35 different SLD devices at varying trap
aspect ratios, main channel height (d.sub.c) to trap height
(d.sub.t) ratios, and trap configurations were fabricated for
testing. For each loading trial a test solution of DI water,
glycerol, blue food coloring (for imaging) and a small
concentration of TritonX100 surfactant (0.06% w/w) was used. The
surfactant was added to aid self-filling. The measured advancing
contact angle of the sample solution was 90.degree..
[0095] In yet another embodiment, each of the 35 SLD chips had a
w.sub.c=200 .mu.m and w.sub.t=390 .mu.m because those are the
smallest dimensions that could be repeatedly produced at a high
quality with computer numerical control (CNC) machining.
Additionally, the ratio of the main channel height to the trap
height was varied to explore the effects of improved capillarity in
the traps during loading. The total amount of traps on each chip
was 15 for SS, 30 for DS, and 29 for S configurations so that all
traps could be visible within the field of view of the microscope.
Each chip was loaded with 2 .mu.L of test solution and then
subsequently examined under microscope to determine the amount of
traps successfully loaded. The SLD traps ability to load sample
solution into them without trapping air, or its loading percentage,
was the performance metric that each design was evaluated by. More
specifically, the fraction of traps that were completely filled
with sample over the total number of traps in the device was
recorded for 5 trials per device with the loading percentage being
the mean of those 5 trials. The trapping of air bubbles is of
particular concern for digitization applications where polymerase
chain reaction (PCR) is employed because adverse effects of thermal
expansion in a closed microfluidic system. Furthermore, incomplete
filling makes it unsuitable for applications where uniform aliquots
are needed for proper quantification, such as in digital qPCR. The
loading percentage results as demonstrated in FIG. 5 show as
predicted by the numeric model that there is a significant
difference in trap loading percentage for each configuration as the
TAR exceeds 1. The staggered configuration maintains a high loading
percentage at TARs>1, showing that even with non-ideal
conditions the pining effect is significant.
[0096] FIGS. 6A-6C illustrate the design space for the single
sided, double sided, and the staggered configurations,
respectively. The geometric model trap aspect ratio threshold is
denoted by a black dashed line with the area shaded in green
representing the parametric combinations the model predicts will
have a high loading percentage. Data points for each parametric
combination are plotted and connected by a solid black line and its
shadow is color coded for each FAR/POR. The data points that lie
beyond the model threshold are shaded with a darker color and
correspondingly show a sharp drop-off in loading percentage while
the more transparent shadow represents data that falls in the
region of expected high loading percentage.
[0097] In one embodiment, 46 different SLDs chips (15 S, 15 SS, and
16 DS) were tested to assess loading percentage parametrically. SLD
devices with FARs of 0.01, 0.26, 0.51, and 1.00 for DS and SS
configurations, and a FAR of 0.55 with POR of 0.01, 0.13, 0.26,
0.51, and 1.00 for the staggered configuration were fabricated and
tested using the same loading protocol. FIGS. 6A-6C show the design
space for the single sided (FIG. 6A), double sided (FIG. 6B), and
staggered (FIG. 6C) configurations. Each model shows a gradient in
loading percentage decay in the vicinity of the limit. It also
maintains that despite non-ideal conditions the staggered design
will have significantly better loading percentage at TAR>1 which
decays as the POR increases.
[0098] FIG. 7A is an image taken of an SLD chip array 700 after
loading operations. In one embodiment, the SLD chip array 700 has
768 traps 104 loaded with sample and backfilled with oil to isolate
the trapes 104. To demonstrate the scalability of the SLD chip, a
768 trap array was fabricated with 6 parallel channels, each
comprising of 128 traps 104. The robust filling mechanism of the
SLD chip provides it with the flexibility to design any combination
of parallel main channels with any number of in series traps
depending on the specific assay/application and time
constraints.
[0099] In one exemplary embodiment, traps 104 in the array were
designed to have a trap aspect ratio of 1.1 and held approximately
11 nL of sample when completely filled. To load the device, 2.1
.mu.L sample solution was introduced into each parallel channel
which loaded 128 traps plus a sacrificial 129th trap in
approximately 1.5 min. The 129th trap served as a sacrificial trap
so that the 128th trap would have a pining point. During loading
765 out of 768 traps were loaded (99.6%) without trapping air
following closely to the aforementioned model for the device
design. In total, 12.6 .mu.L of sample solution was used to load
the whole device in approximately 5 min with an estimated sample
retention of 60%.
[0100] FIG. 7B shows a histogram of the trapped volumes in the SLD
array. A MATLAB image analysis script was used on a composite image
of the array for acquiring area measurements or each trap based on
a pixel count. A standard deviation of 1.36 (nL) in volume captured
for each trap was measured.
[0101] FIG. 7C illustrates a self-loading chip according to another
embodiment of the present invention. The self-loading chip
comprises a sample inlet port 702, an oil inlet port 704, an outlet
port 706, a sample inlet channel 710, an oil backfill channel 708,
and sample traps 712. The sample traps 712 are in fluid
communication with both the sample inlet channel 710 and the oil
backfill channel 708. Each trap is connected to the oil backfill
channel 708 through a high .beta.-angle hydrophobic valve to
prevent sample from leaking from the trap chamber 712 into the
backfill channel 708 during sample filling. Furthermore, each trap
712 comprises lyophilized reagents. In one embodiment, the
lyophilized reagents include primers for performing a nucleic acid
amplification reaction. The pre-deposited reagents may be
loop-mediated isothermal amplification (LAMP) reagents, Polymerase
Chain Reaction (PCR) reagents, Nucleic acid sequence based
amplification (NASBA) reagents, Helicase-dependent Amplification
(HAD) reagents, Strand Displacement Amplification (SDA) reagents,
Recombinase polymerase amplification (RPA) reagents, Hybridization
Chain Reaction (HCR) reagents, or Rolling Circle Amplification
(RCA) reagents including primers to amplify target nucleic acids in
the sample.
Exemplary Embodiment 1. Chip Fabrication
[0102] In one aspect of the present invention as shown in FIG. 8B,
a self-loading and digitizing (SLD) device such as the chip 800
demonstrated in FIG. 8A was fabricated from a thermoplastic. In one
embodiment, COP was used due to its high transparency, low
autofluorescence, low water absorption, and low gas permeability,
making it a suitable substrate material for PCR. The microfluidic
features can be created by any number of ways including single-step
embossing (if all features are the same height), two-step embossing
(for different heights), milling, as well as injection molding and
other common techniques for thermoplastic patterning.
[0103] Specifically, in the embodiment of FIG. 8B, a channel layer
824 was fabricated from a thermoplastic substrate in a two-step
embossing process (step 802). In one embodiment, a mold 824 was
fabricated using CNC machining to create a plurality microfluidic
features including a microfluidic channel 102, sample traps 104,
inlet/outlet ports 112/116, and a capillary pump chamber 106. Then,
a thermoplastic substrate 826 was pressed against the mold 824. In
step 804, the substrate is solvent exposed. Parallel with the
substrate fabrication of steps 802 and 804, reagents 820 are pin
spotted on a sealing layer 818 in step 808. In one embodiment, the
regents include primers in sucrose. Then, the reagents were
lyophilized in step 810. The channel layer 826 was also milled with
alignment holes 816 in each corner so that it could be aligned to
protruding pins on the pin spotting stage 818 that would keep it
aligned with the same origin on the pin spotter each time (step
806).
[0104] In one embodiment, two PVDF membranes were patterned using a
craft cutter and were then inserted into a pre-milled chamber 106
on the channel layer 826. A pointed shape at the front of membrane
provided the best performance in terms of purging excess sample
from the main channel. The milled substrate was then mated to the
thin film COP with deposited reagents 820 face down on the spotting
stage 818. Tape on the COP film was wrapped around the substrate on
top to hold it together until the appropriate pressure applied was
used to complete bonding in step 806. Electrode photolithography
was performed in step 812, followed by Au deposition and liftoff in
step 814.
[0105] In one exemplary embodiment, the channel layer of the SLD
chip as shown in FIG. 8A was fabricated by milling channel and
microwell features in a 2 mm thick COP plaque (by way of example
and without limitation, Zeonor 1020, Zeon Chemicals, Louisville,
Ky.) to a depth of 250 .mu.m using a 3-axis CNC machine (by way of
example and without limitation, MDX-650, Roland DGA, Irvine,
Calif.). A 125 .mu.m thick hydrophilically modified polyvinylidene
fluoride (PVDF) membrane with 5 .mu.m pore size (by way of example
and without limitation, SVLP04700, EMD Millipore, New Bedford,
Mass.) was patterned using an automated craft cutter (by way of
example and without limitation, Cameo Digital Craft Cutting Tool,
Silhouette America, Orem, Utah). The capping layer of the SLD chip
was patterned from a 50 .mu.m thick cyclic olefin copolymer (COC)
film (by way of example and without limitation, Zeonex 1420, Zeon
Chemicals). The COC film was affixed to the pin spotting stage with
tape to prevent movement. Primer solution consisting of 20% (w/w)
polyethylene glycol (PEG) was loaded into the pin using capillary
action and then deposited when the pin made contact with the film.
After deposition the PEG/primer spots were left to completely dry
at 45.degree. C. for 15 min before a protective layer of soft
paraffin wax (by way of example and without limitation, Vaseline,
Unilever, USA) was deposited at a temperature 42.degree. C. over
top of the dried PEG spots. The microchannel substrate was next
exposed to 35% (w/w) decalin (by way of example and without
limitation, ThermoFisher Scientific, Rockford, Ill.) in ethanol for
1.5 min, rinsed with 100% ethanol, and blown dry with N.sub.2. Two
identically patterned PVDF membranes were stacked for a total
thickness of 250 .mu.m and inserted into a mating chamber in the
COP substrate. The decalin-solvated channel substrate and capping
layer were then aligned using pins built into an alignment stage
and mated to seal the channels and microwells. The chip assembly
was then placed in a hot press (by way of example and without
limitation, AutoFour/15, Carver Inc., Wabash, Ind.) at 200 psi and
23.degree. C. for 10 min to complete the bonding.
[0106] In yet another exemplary embodiment, a custom pin spotting
tool was provided for the controlled deposition of PCR reagents
onto COP films. The tool consisted of three linear actuators (by
way of example and without limitation, MX45S, Parker Hannifin
Corp., Cleveland, Ohio) attached to stepper motors (by way of
example and without limitation, LV141-02-10, Parker Hannifin Corp.,
Cleveland, Ohio) for X, Y, and Z axis control. Photoelectric
sensors with 30 .mu.m repeatability (by way of example and without
limitation, PMY44P, Parker Hannifin Corp., Cleveland, Ohio) were
used to calibrate the origin and provide limit stops to protect the
actuators from damage. The motors were driven by a motor driver (by
way of example and without limitation, ED-Drive, Parker Hannifin
Corp., Cleveland, Ohio) enabling positioning resolution of 1 .mu.m,
and controlled using an Arduino Uno microcontroller (by way of
example and without limitation, Adafruit, New York, N.Y.) and GRBL
open source software for the graphical interface. The Z-axis
actuator controlled the height of various sized pins (by way of
example and without limitation, Xtend Microarray Pin, LabNext,
Inc., West New York, NJ) that deposited nanoliter scale volumes
through contact printing. The spotting stage was equipped with
mechanical alignment pins for gross positioning of the channel
layer to the spotter. The stage was also equipped with two Peltier
heaters; one positioned under the spotting film, and one in a
peripheral location to heat the wax bath.
Exemplary Embodiment 2. Chip Fabrication
[0107] The SLD devices according to FIG. 1A and FIG. 7A were
fabricated by milling channel and microwell features in a COP
plaque (by way of example and without limitation, a 2 mm thick
plaque, Zeonor 1020, Zeon Chemicals, Louisville, Ky.) using a
computer numerical controlled 3-axis CNC machine (by way of example
and without limitation MDX-650, Roland DGA, Irvine, Calif.). A
hydrophilically modified polyvinylidene fluoride (PVDF) absorbent
membrane (by way of example and without limitation, SVL04700, EMD
Millipore, New Bedford, Mass.) was cut to specific size using an
automated craft cutter (by way of example and without limitation,
Cameo Digital Craft Cutting Tool, Silhouette America, Orem, Utah).
The milled COP plaque was immersed in a solution of 35%
decahydronaphthalene (by way of example and without limitation,
Thermo Fisher Scientific, Rockford, Ill.) in ethanol (w/w) for 1.5
min, rinsed with ethanol, and blown dry with N.sub.2. The absorbent
membrane was then manually aligned to a premilled chamber in the
COP plaque before the multilayer device was pressed at 200 psi and
23.degree. C. for 10 min in a hot press (by way of example and
without limitation, AutoFour/15, Carver Inc., Wabash, Ind.) to
complete the bonding.
Exemplary Embodiment 3. Self-Loading and Digitization Operation
[0108] Loading experiments for all SLD devices according to the
present invention were conducted by using a pipette to manually
load 2 .mu.L of DI water containing 0.06% (w/w) TritonX100 (Sigma
Aldrich), glycerol, and blue food coloring. Once the sample primed
the device and the absorbent membrane removed excess sample, the
chip was imaged under the microscope (by way of example and without
limitation, AZ100, Nikon Instruments Lewisville, Tex.) to identify
the number of traps that had been successfully loaded. The trapping
of small air bubbles or incomplete loading of a well would be
considered unsuccessful. Priming and purging was accomplished in
approximately 30 s for devices with 30 or fewer traps, while the
higher density device was primed and purged within approximately 5
min.
[0109] An SLD devise according to one embodiment of the present
invention is provided to perform highly scalable multiplexed PCR
with minimal manual input. Specifically, FIG. 8A illustrates a
thermoplastic self-loading chip 800 comprising an inlet port 116,
an outlet port 112, a capillary pump 106, a microchannel 102. The
microchannel 102 connects the inlet port 116 to the capillary pump
806 at the outlet 112 with staggered wells (traps) 104 in between.
The array of microwells 104 serves to isolate discrete sample
volumes, while PCR reagents integrated into the chip during
fabrication allow for different reactions to be performed within
each discretized volume.
[0110] Reagents are integrated into the wells by pin spotting
sequence-specific PCR primers in a paraffin wax matrix, ensuring
that the primers remain encapsulated during sample introduction
while enabling temperature-controlled release prior to
thermocycling. In one embodiment, the reagents are lyophilized. In
yet another embodiment, reagents may be deposited by piezoelectric
inkjet, screen printing, and solid-phase deposition.
[0111] The sample itself is manually deposited by pipette into the
inlet port 116, with passive filling and discretization of the
entire microwell array achieved by capillary pumping as shown in
FIG. 1A. Effective self-filling of the thermoplastic reaction
chambers is achieved through the use of a staggered array design
that employs geometric fluid pinning to promote highly repeatable
filling of the high aspect ratio microwells (traps). In one
embodiment, the capillary pump 106 is a PVDF membrane.
[0112] In one exemplary embodiment, microwell dimensions of 900
.mu.m square and 250 .mu.m deep were chosen to accommodate the
paraffin wax covered reagents and provide sufficient reaction
volume for effective PCR from dilute samples while still providing
reliable self-filling and digitization.
[0113] In one embodiment, chip loading was performed by pipetting
sample solution in the inlet 116 of the chip 800. Excess sample was
removed by the integrated PVDF membrane 106 downstream and then an
immiscible phase was loaded into the chip 800 to fully isolate each
reaction well (trap). Once loaded, a single piece of PCR-compatible
adhesive tape was used to seal the top side of the chip. The chip
was then placed on a thermocycler comprising a Peltier element
controlled by a microcontroller. The software-defined PCR routine
implemented by the microcontroller was performed. The
microcontroller actuated a LED light source (not shown) and a CCD
camera (not shown) placed directly above the chip to collect
fluorescence output during the extension step of the PCR
reaction.
[0114] In one exemplary embodiment, 3.75 .mu.L of sample solution
was pipetted into the inlet port 116 of the chip 800. Excess sample
was removed by the integrated PVDF membrane 806 downstream and then
5 .mu.L silicone oil (for example, AR20, Sigma-Aldrich, St. Louis,
Mo.) was loaded. The PCR reaction consisted of a 120 s hot start at
95.degree. C., followed by 20 cycles of 95.degree. C. for 15 s,
60.degree. C. and 72.degree. C. for 30 s. The microcontroller
actuated the 452 nm LED light source and a CCD camera collected
fluorescence output during the extension step (72.degree. C.).
[0115] Preventing the rehydration of integrated primers during
sample introduction is essential to the proper functioning of the
SLD chip. Without slowing rehydration, primers will be carried by
the fluid front and transported downstream cross contaminating
subsequent sample traps.
[0116] In one embodiment, polysaccharides were selected for a
dissolution retarding matrix for the integrated primers.
Specifically, sucrose, dextran, and polyethylene glycol (PEG) were
selected for their solubility in water, and general
biocompatibility with polymerase chain reaction. Each was dissolved
at concentrations ranging 10% to 40% (w/w) and mixed with
fluorescein salt. Specifically, in one exemplary embodiment, a
concentration of 20% (w/w) for each polymer was found to
successfully balance viscosity appropriate for deposition and
dissolution time. The different mixtures were spotted on the COP
substrate and dried down. A droplet of water with a comparatively
large volume was added to the spot and fluorescence was recorded
over time. In this way the approximate dissolution time (diffusion
limited) for each additive was determined.
[0117] Accordingly, sucrose, dextran, and PEG spots were
incorporated into an SLD device. Then, a sample solution was
self-loaded to confirm the effect of advective flow in the sample
traps. Fluorescence in the traps was recorded over time to
determine the approximate dissolution time with the addition of
advection. FIG. 9 shows fluorescence images over time from PEG 20%
(w/w) and fluorescein salt spots as sample solution is self-loading
through the chip as shown in FIG. 8A.
[0118] In yet another embodiment, gelatin was tested to retain
primers to the trap during sample loading. Gelatin improved
diffusion limited transport and could fully expel the incorporated
dye when heated above its melting point, however during device
testing once the gel was hydrated advection would dominate
dissolution and disperse the fluorescein salt at a similar time
scale to sucrose and PEG.
[0119] In one embodiment, to mitigate the effects of advection,
paraffin wax, a hydrophobic material containing hydrocarbons of the
general formula C.sub.nH.sub.2n+2, was used. Paraffin wax has been
shown to be both compatible with PCR and able to protect dried
reagents against flow. A robust coating of paraffin overtop of a
primer spot provides an indefinite protective barrier to primer
dissolution. The paraffin could be heated to above its melting
point after sample solution was loaded to disperse the primers into
corresponding sample traps (reaction chamber). Table 1 summarizes
the spotting and dissolution data for the mentioned additives.
TABLE-US-00001 TABLE 1 Summary of primer additive materials for
reagent integration DISSOLUTION TIME DISSOLUTION TIME MATERIAL
SPOTTING VARIABILITY CONTROLLED RELEASE (DIFFUSION) (DIFFUSION +
ADVECTION) Sucrose (20%) .sigma. = 5.9% No 20 s 2 s (MW = 310 Da)
PEG (20%) .sigma. = 1.8% No 60 s 8 s (MW = 20,000 Da) Dextran (20%)
.sigma. = 14.3% No 90 s *N/A (MW = 70,000 Da) Gelatin .sigma. =
2.0% **Yes (T > 60 C.) 200+ s 5 s (300 bloom) Paraffin Wax
.sigma. = 2.5% Yes (T > 60 C.) Indefinite Indefinite Note:
*denotes that test was not attempted because spotting was too
inconsistent a. **denotes that controlled release was in the
diffusion limited case only
[0120] Temperature control was an integral factor in successfully
spotting the paraffin wax. In one exemplary embodiment, a
sufficiently thick layer of paraffin (>30 .mu.ms) was required
to completely protect the primers underneath during sample loading.
To achieve sufficient thickness, the temperature had to be kept
above the paraffin melting temperature of 37.degree. C. to keep the
solution viscous, but below 50.degree. C. where the paraffin
solution would easily wet out on the surface of the COP spreading
the wax layer outside the bounds of the traps compromising the
surrounding bonding surface. The pin has to be completely free
floating or damage can occur during contact with the stage. In one
embodiment, a "pick and place" technique where a solid pin would be
dipped in a hot wax bath (by way of example and without limitation,
T=100.degree. C.) and when retracted would retain a dollop of wax,
slightly larger than the pins outer diameter. The pin would then
make contact with the substrate held at 42.degree. C. (closely
above the wax melting temp of 37.degree. C.) and the wax would melt
with a viscosity that resulted in a sufficiently think mound
covering the primer spot.
Reagent Integration and Controlled Release
[0121] In one embodiment, PEG was included as an additive to the
primer solution. By way of example and without limitation, PEG
concentration may be 20% (w/w). The PEG served to improve the long
term stability of the dried down oligomers as well as increase the
viscosity of the spotting solution, reducing spot variability.
Additionally, when dried down, the PEG additive crystallized into a
solid which served as a way of visualizing the primers so that
proper alignment with the subsequent wax deposition could be easily
characterized. In one exemplary embodiment, as illustrated in FIG.
10A, using a 300 .mu.m diameter pin tip with PEG 20% (w/w) mixed
with primer resulted in a nominal spot size of 113.5 .mu.m
(standard deviation .sigma.=2.1 .mu.m). The spot 1002 had a contact
angle of 45.degree. with the COP film resulting in an estimated
deposited volume of approximately 0.2 nL.
[0122] Paraffin wax was used as a capping layer 1004 for the
PEG/primer spot. Using a 200 .mu.m diameter pin resulted in a
nominal paraffin wax spot size of 249.4 .mu.m (standard deviation
.sigma.=6.3 .mu.m) as shown in FIG. 10B. As demonstrated in FIG.
10C, paraffin capped primers were spotted into the upper center
1006 of the traps because that was the center of the reaction
volume that was captured during loading. The paraffin provided a
robust protective layer overtop the primers preventing rehydration
during several repeated flushes with Methanol, Isopropanol and DI
water though the device.
[0123] FIG. 11 illustrates release of the incorporated primers by
adding fluorescein salt to the primer solution. The chip was loaded
and backfilled with oil with no measurable difference in the
fluorescence of the spots. Images were then taken as the chip was
heated up to 70.degree. C. showing the fluorescence dispersing from
the original spots as the temperature reaches .about.65.degree.
C.
[0124] Proper self-loading of the SLD chip, as shown in FIG. 8A,
relies on the surface interactions of the sample solution and the
chip substrate. A model of surface tension driven trap loading was
discussed with reference to FIGS. 2A-2D, 2F, 3A-3D, 3F, and 4A-4D,
4F. However, in order to integrate PCR reagents into the sample
traps, a hydrophobic material (paraffin wax) is deposited on the
surface of the traps resulting in a deviation in loading percentage
from the results as discussed above with reference to FIGS. 2A-2D,
2F, 3A-3D, 3F, and 4A-4D, 4F. After incorporating the paraffin
spots, aqueous sample solution would circumvent the hydrophobic wax
surface and leave behind an air bubble in the traps over the
paraffin mounds. To overcome this deficiency, the depth of the
channels was increased such that a lesser pressure change would be
required to overcome the hydrophobic wax mound. Trap depth had to
be limited however because self-loading and sheer induced
separation during purging would be negatively impacted by
increasing the depth of the channel. In one embodiment, a trap
depth of 250 .mu.m was found to be the optimum for reliable bubble
free loading while also providing sufficient sample retention
during purging. Trap dimensions of 900 .mu.m (wide).times.900 .mu.m
(long).times.250 .mu.m (deep) resulted in a mean captured volume of
92 nL (RSD=9.2%). Digitized volume was extrapolated by using image
analysis to measure the surface area of the captured sample and
multiplying it by the depth of the trap.
Multiplex PCR
[0125] The ability to detect different template DNA based on the
specific integrated primer sets was important in proving the
utility of the SLD chip as discussed above with reference to FIGS.
1A, 7A, and 8A for potential use in bacterial species
identification and antibiotic resistance screening. Primer sets
were designed to demonstrate two scenarios: 1) primer sets
compatible with template would amplify while non-compatible primers
would not, 2) if both primer sets would amplify with the loaded
template then their respective melt temperatures (Tm) could be
distinguished during an on-chip high resolution melting analysis
(HRMA). To accomplish the first scenario two primer sets p19 and
p322 were designed such that the p19 primer set would amplify
plasmid pUC19 template and the p322 primer set would not. For the
second scenario it was insured that p19 would also amplify with
plasmid pBR322 template so when loading pBR322 template both p19
and p322 primer sets would be amplified, but yield amplicons with
differing melt temperatures.
[0126] In one exemplary embodiment, the pUC19 and pBR322 plasmids
were diluted to 30 ng/.mu.L. Each template was mixed with equal
volumes of commercial master mix and EDTA buffer to form sample
solution with a final concentration of 10 ng/.mu.L, or
approximately 2.0 ng per reaction. The mastermix included LCGreen,
a DNA-intercalating dye, to enable on-chip fluorescence detection
of PCR reaction product and high resolution melt analysis (HRMA)
for product validation. The pUC19 forward primer
(5'-GACCTACACCGAACTGAGATACC-3') (SEQ ID NO: 1) and reverse primer
(5'-TCCGACCCTGCCGCTTAC-3') (SEQ ID NO: 2) as well as pBR322 forward
primer (5'-TGCTCAACGGCCTCAACCTA-3') (SEQ ID NO: 3) and reverse
primer (5'-AGTCATAAGTGCGGCGACGA-3') (SEQ ID NO: 4) were designed.
In one embodiment, Primer3Plus software was used to design the
primers. Both primer sets were diluted to form stock solutions at 5
mM. The primer printing solution contained 10.times.5 mM primer
stock, 2.times. buffer (Novella Oligo Dilution Buffer, Canon US
Life Sciences, Rockville, Md.) and 3.times.50% PEG solution, for a
final primer concentration of 500 .mu.M in the reaction volume.
[0127] FIG. 12A is an illustration summarizing the expected
experimental outcomes. The p19 primers and pBR322 template produced
an amplicon with similar Tm as the p19 primer and pUC19 template.
In one embodiment, both primer sets were designed to amplify under
the same thermocycling conditions--20 cycles of 95.degree. C. for
15 s, 60.degree. C. and 72.degree. C. for 30 s. By way of example
and without limitation, the assay was run on a Roche LC480
beforehand and HRMA confirmed that the p19 primers and pBR322
template reaction yielded amplicons that had a Tm 2.3.degree. C.
lower than the amplicons produced by p322 primers and pBR322
template.
[0128] The SLD chip was designed to minimize workflow such that
only two pipetting steps (one for sample and one for oil) is all
that is required to discretize a sample into separate reactions.
This was realized experimentally with a 16 trap device loaded in
less than 60 s. A trap loading percentage of 75% and 100% for pUC19
template and pBR322 template loaded chips, respectively, was
achieved. HRMA showed that the traps filled with p19 produced an
amplicon with a mean Tm=86.4.degree. C. (standard deviation
.sigma.=0.3.degree. C.). The chip loaded with pBR322 template
loaded all traps successfully. Traps filled with the p19 primers
amplified a product with a mean Tm=87.3.degree. C. (standard
deviation .sigma.=0.7.degree. C.), and traps filled with the p322
primers produced an amplicon with a mean Tm=89.5.degree. C.
(standard deviation .sigma.=0.7.degree. C.). A difference in the Tm
of 2.2.degree. C. between p19/pBR322 and p322/pBR322 amplicons is
in good agreement with the difference in amplicon Tm measured on
the LC480.
[0129] The need for sample preparation presents a significant
challenge toward any nucleic acid diagnostic designed for use at
the point-of-care. In one embodiment of the present invention,
sample preparation is decoupled from the microfluidic amplification
and SLD detection platform, allowing the process of nucleic acid
extraction, purification, and concentration to be modified for
specific sample types while maintaining a universal microfluidic
platform for back-end analysis. Specifically, the sample
preparation is performed using functionalized pipette tips. An
example of workflow is depicted in FIGS. 13A-13C, where the custom
pipette tip takes advantage of chitosan functionalization of porous
polymer monoliths used for DNA capture and release.
[0130] As depicted in FIG. 13A, raw sample is drawn through a
chitosan monolith, where charge interactions capture free nucleic
acids on the porous surface. A pre-filter element integrated into
the tip also serves to remove particles and cell debris from the
sample. In one embodiment, the pre-filter element is a chitosan
monolith 1302. Specifically, 1 mm thick chitosan monolith element
1302 is sufficient to capture DNA with loading levels over 100 ng
per square millimeter of monolith area. After capture, the sample
is washed with pH 5 buffer to remove contaminants (FIG. 13B), after
which amplification buffer at pH 8.8 was loaded into the pipette
and used to elute concentrated DNA directly into the trap chip
(FIG. 13C). In yet another embodiment, this process is simplified
by using a syringe rather than a pipette, with rinse and elution
buffers preloaded into the syringe body.
[0131] Accordingly, in addition to decoupling sample preparation
and amplification in a process that is tractable for use at the
point of care, this approach also eliminates the need for
chaotropic salts for DNA concentration and purification, solves the
world-to-chip interface challenge, and enables reliable control
over sample volume during nucleic acid elution without the need for
complex or bulky instrumentation.
[0132] Multiplexed assays using nucleic acid amplification offer
particular potential in point-of-care settings where the ability to
rapidly detect multiple biomarkers with high specificity and
sensitivity can have a transformative effect on healthcare.
However, established PCR-based nucleic acid diagnostics require
significant instrumentation to support multiplexing, making them
too cumbersome, costly, and difficult to use at the point of care.
Furthermore, conventional PCR is too slow for many point-of-care
applications.
[0133] According to one aspect of the present invention, a
microfluidic platform is provided to overcome these limitations and
use multiplexed nucleic acid diagnostics in point-of-care settings.
The platform employs a thermoplastic chip designed to automatically
segregate an initial sample volume into large numbers of isolated
reaction chambers containing all reagents necessary for multiplexed
amplification and detection, without the need for any external
pumps or valves, substrate preparation, or reagent introduction.
Simultaneous on-chip amplification is achieved by using
loop-mediated isothermal amplification (LAMP), greatly simplifying
the system-level multiplexing requirements. Significantly, through
the use of contact imaging, the system is exceptionally compact,
ultimately allowing all functions to be integrated into a USB stick
format that supports assay operation and readout using a notebook
or tablet computer.
[0134] LAMP operates at 60-65.degree. C. and exponentially
amplifies DNA sequences through the formation of loops in the
amplicons, enabling primer binding without DNA melting. In addition
to exponential amplification, the method is rapid because of the
elimination of the thermal cycling time as well as the continued
evolution of the Bst polymerase.
[0135] Amplification reactions can reach saturation in as fast as
ten minutes. For increased specificity, the LAMP reaction (Tanner
et al., "Simultaneous multiple target detection in real-time
loop-mediated isothermal amplification," Biotechniques, vol. 53,
no. 2, pp. 81-89, 2012) is demonstrated in FIG. 16. Following the
initial melt step, the primers F2 and F3 bind to the target, step
1602. F2 has attached to it F1c, which is hybridized with FD; FD
contains a donor fluorophore while F1c contains a quencher. In step
1604, polymerase binding at F2 will extend a complement to the
template, while polymerase that binds at F3 will also extend the
template while displacing the other strand. As the strand is
displaced, a loop is formed by the self-hybridization at F1; this
loop formation also displaces FD, which leads to an increase in
fluorescence, step 1606. Next, a similar process occurs in the
reverse direction at B1c, B2c, and B3c, step 1608. Ultimately, this
first round of copying generates strands with loops on each end,
step 1610. The loops enable primer access without further DNA
melts, thus leading to the repeated copying of these loop-based
structures at an exponentially increasing rate. Signal is also
generated at an exponentially increasing rate due to the continued
displacement of FD strands at each copying of the loop
structures.
[0136] In one exemplarily embodiment, a disposable thermoplastic
microfluidic chip supporting multiplexed LAMP assays is fabricated
in COP substrate with a high density array of up to 1024 reaction
chambers (by way of example and without limitation, 32.times.32
wells, 100 nL volume) in a footprint below 2.5 cm.sup.2.
Lyophilized LAMP reagents, including Bst polymerase and
target-specific primers, are integrated on-chip by microarray
spotting in a wax matrix to enable controlled release of reagents.
Thin film platinum heaters and thermistors supporting precise
temperature control are patterned on the chip surface.
[0137] Accordingly, a simple, automated, and disposable platform
capable of rapidly amplifying up to 1024 independent gene targets
using spatially multiplexed isothermal amplification is uniquely
suited for near-patient settings.
[0138] A key advance of the present invention is the development of
on-chip multiplexed LAMP as an isothermal amplification technique.
In contrast to PCR, LAMP reactions are performed at a constant
temperature, eliminating the need for rapid thermocycling. Thus,
moving to isothermal amplification greatly simplifies system-level
operation of the devices to allow for USB integration. The lower
temperatures associated with LAMP amplification (typically
60-65.degree. C.) serves to reduce the impact of thermally-induced
bubble formation. Furthermore, because each LAMP reaction employs
multiple primers to identify distinct regions on the target gene,
specificity is greatly enhanced over PCR, while the addition of
loop primers accelerates the reaction, allowing amplicons to be
generated at a higher rate than PCR for assay times.
[0139] While the staggered trap array, as demonstrated in FIG. 7A,
successfully enabled automated sample filling and discretized PCR,
progress was limited due to fabrication challenges relating to chip
bonding. In one aspect of the present invention, a new array design
in which the sample traps are fabricated as vertical wells beneath
a planar microchannel, rather than lateral protrusions emerging
from the sides of a single linear microchannel. FIGS. 15A-15B
demonstrate comparison of the staggered trap design (FIG. 15A) to
the planar vertical trap design (FIG. 15B). Each chip, as shown in
FIGS. 15A-15B, comprises a sample inlet 116, a filling channel 102,
a trap array 104, and a capillary pump 106.
[0140] Initial state 1502 demonstrates two trap array
configurations prior to receiving the sample at the sample inlet
116. The staggered trap design is shown in FIG. 15A and the planar
vertical trap design is shown in FIG. 15B. Each sample trap (well)
104 contains LAMP reagents 1510 for performing an amplification
reaction. Cr/Au electrodes 1512 are attached to the chip. A sample
is introduced through the sample inlet port 116 and fills the
sample traps 104, step 1504. After an excess sample has been
removed by the integrated capillary pump (PVDF membrane) 106, the
oil is introduced into the channel 102 to isolate the sample traps,
step 1506. In step 1508, Cr/Au electrodes 1512 provide for
heat-assisted release of LAMP reagents to amplify target nucleic
acids in the sample.
[0141] Vertical trap filling process is demonstrated in FIG. 14. In
one embodiment, effective loading of square traps with in-plane
dimensions ranging from 50 .mu.m to 250 .mu.m and heights ranging
from 50 .mu.m to 100 .mu.m have been successfully demonstrated,
with sample filling and discretization during oil backfill
achieved. Filling of the chips is facilitated by activation of the
thermoplastic surface containing the microwells through exposure to
UV/ozone, generating in a dense layer of negatively charged groups
on the surface, resulting in high surface energy and promoting
hydrophilic anchoring of discrete sample volumes within the traps
during oil backfill. The use of surface activation also
significantly enhances the speed of the filling process over
staggered trap arrays due to higher capillary forces in the
hydrophilic devices. In one embodiment, a staggered trap array
concept may be adapted to the vertical trap design by patterning a
second set of staggered 2D traps on the opposing channel surface to
increase the achievable trap volume.
[0142] Because the wells in the vertical design are located far
from the bond interface, bubble formation does not impact
amplification or assay readout, allowing the use of robust solvent
bonding methods for high chip yield, while the 2-D geometry of the
filling channel allows large arrays of sample traps to be rapidly
(by way of example and without limitation, <30 sec) filled from
a single inlet port. Avoiding the high temperatures necessary in
PCR allows for reducing thermally induced bubble formation.
[0143] To enable multiplexed amplification, target-specific LAMP
primers and polymerase are deposited by mechanical spotting in
individual trap chambers, and controllably released during an
initial LAMP heating step. An effective technique is provided for
depositing PCR primers in a PEG matrix, with paraffin wax
encapsulation supporting thermally-controlled reagent release,
thereby preventing primer cross-talk between traps during sample
filling (FIG. 10A-10C).
[0144] In one exemplarily embodiment, the combination of the planar
vertical trap chip design and reagent spotting allows for achieving
a 32.times.32 array of 1024 traps containing integrated reagents.
Trap chamber has dimensions of 500 .mu.m diameter and 500 .mu.m
height for a well volume of approximately 100 nL, and a total
sample volume of .about.100 .mu.L for the entire array. This array
design resides within a 2.5 cm.sup.2 footprint, allowing optical
detection from the full array using a simple contact imaging system
integrated into a USB stick.
[0145] A reusable CMOS contact fluorescence system 1806 integrated
into a USB stick 1800 along with a trap-array chip 1802 and LED
array 1814 is demonstrated in FIG. 18. The external thermoelectric
(for example a thermocycler) used for temperature control in SLD
chips is replaced with integrated thin film Cr/Au electrodes,
significantly reducing power requirements for the devices.
Specifically, Cr/Au electrodes (element 1512 in FIGS. 15A-15B) are
patterned on the chip backside for temperature sensing and
actuation. The electrodes are positioned in the periphery of each
trap to avoid blocking optical access from either the top or bottom
chip surfaces. All interface electronics is implemented on a single
printed circuit board 1810 using a stamp-sized microcontroller.
[0146] Fluorescence imaging of the trap-array 1802 is performed
using front-side LED illumination 1814 and back-side detection
using the CMOS contact imager 1806, obviating the need for any
optical focusing components in the system. By placing the CMOS
imager 1806 in close proximity to the imaging surface,
high-resolution optical detection can be achieved. An excitation
filter glass 1816 is placed between the LED source 1414 and
trap-array chip 1802, while an emission filter glass 1804 is placed
between the trap-array chip and CMOS detector 1806.
[0147] The realization of a rapid multiplexed diagnostic operated
from a laptop computer, without the need for any external
instrumentation, opens the door to environments ranging from small
clinics, individual doctor offices, remote settings with limited
access to conventional laboratory infrastructure or other
resources, and ultimately in-home use. To this end, a CMOS chip
supporting a standard SPI (I2C) interface can be employed to
simplify USB communication, with Java code on the host computer
used for SPI control and data collection from the imager, together
with control of the LED light sources 1814 and thin film
temperature control elements.
[0148] High fidelity fluorescence images are achieved using a bare
consumer-grade CMOS imager chip as shown in FIGS. 17A-17B. FIG. 17A
demonstrates a bare CMOS imager positioned below a trap chip. FIG.
17B demonstrates contact imaging results using fluorescent dye.
Fluorescence signal detected at each trap 1704 and the channel 1702
is filled with oil are shown in FIG. 17B.
[0149] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0150] While the subject matter of this disclosure has been
described and shown in considerable detail with reference to
certain illustrative embodiments, including various combinations
and sub-combinations of features, those skilled in the art will
readily appreciate other embodiments and variations and
modifications thereof as encompassed within the scope of the
present disclosure. Moreover, the descriptions of such embodiments,
combinations, and sub-combinations is not intended to convey that
the claimed subject matter requires features or combinations of
features other than those expressly recited in the claims.
Accordingly, the scope of this disclosure is intended to include
all modifications and variations encompassed within the spirit and
scope of the following appended claims.
Sequence CWU 1
1
4123DNAArtificial SequencepUC19 forward primer 1gacctacacc
gaactgagat acc 23218DNAArtificial SequencepUC19 reverse primer
2tccgaccctg ccgcttac 18320DNAArtificial SequencepBR322 forward
primer 3tgctcaacgg cctcaaccta 20420DNAArtificial SequencepBR322
reverse primer 4agtcataagt gcggcgacga 20
* * * * *